Increased inactivation of drugs due to overexpression of the Pi class glutathione S-transferase isoenzyme (hGSTP1-1), which is polymorphic in human populations and involves amino acid residues in positions 104 (isoleucine or valine) and 113 (alanine or valine), is believed to be an important mechanism in tumor cell to alkylating agents. Recent studies from our laboratory indicate that the I104,A113 isoform of hGSTP1-1 is significantly more efficient than the other variants of hGSTP1-1 in catalyzing the GSH conjugation (detoxification) of chlorambucil (CMB) and thiotepa (TT). These observations led us to hypothesize that hGSTP1-1(I104,A113) isoform expressing cells/tumors are likely to be more resistant to the cytotoxic effects of some alkylating agents than cells/tumors containing other variants of hGSTP1-1. The overall objective of this research project is to test the above hypothesis by a series of preclinical studies. In specific aim 1, we will compare the catalytic efficiencies of the four allelic variants of hGSTP1-1 in the GSH conjugation of activated metabolites of cyclophosphamide (CP) and ifosfamide (IF). These studies will reveal whether the hGSTP1-1(IA) isoform is more efficient than other variants of hGSTP1-1 in the detoxification of alkylating agents other than thiotepa and chlorambucil. In specific aim 2, we will investigate the effects of the overexpression of hGSTP1-1 variants, through stable transfection in cultured cells, on the cytotoxic and DNA cross-linking activities of CMB, TT, CP and IF. In specific aim 3, we will determine the correlation between the expression of hGSTP1-1 variants and antitumor activity of the above alkylating agents in vivo. This goal will be achieved by determining antitumor activities of the alkylating agents against hGSTP1-1 variant- transfected cells implanted in nude mice. Studies in aims 2 and 3 will reveal whether the hGSTP1-1(IA) isoform overexpressing cells/tumors are more resistant to the cytotoxic effects of alkylating agents than cells/tumors with the overexpression of other variants of hGSTP1-1. Finally, specific aim 4 is to elucidate the molecular mechanism of differences in kinetic properties of the hGSTP1-1 variants in the GSH conjugation of thiotepa and chlorambucil by X-ray crystallography. In summary, the studies proposed in this application will shed light on the physiological significance of hGSTP1-1 polymorphism in relation to alkylating agent resistance. These studies, in the long term, may be valuable in identifying subjects that may be at a higher risk for developing GST-mediated resistance to some alkylating agents.